Tory Eurosceptic MPs risk rupturing the coalition with a bid to force a vote on exiting the EU.
A group of rebel MPs have tabled an amendment in the House of Commons on holding an in/out referendum within the next 12 months.
Prime Minister David Cameron had promised one in 2017 - after the next election. But a collection of MPs have joined the growing clamour on Conservative benches - by demanding one now.
Rebel Tories ambushed the motion welcoming the Queen's Speech on Wednesday.
Coalition partners the Liberal Democrats would oppose on principle any move towards an EU exit.
Cameron has made it clear this means he cannot side with his own MPs. Among the rebels who signed the amendment was Nadine Dorries, the Mid-Bedfordshire MP who was suspended by the Tories for taking unscheduled leave to take part in I'm a Celebrity and has only just returned to the party.
If enough MPs sign up to trigger a debate and then a vote, it means Liberal Democrat MPs could further rattle the coalition by voting with Labour against the Conservatives.
The row comes a week on from big gains made by anti-EU party Ukip at county elections - traditionally Tory heartlands.
Dorries told the BBC: "We should have a referendum as soon as possible. People were given a clear choice when we went in and it should a clear choice now. The amendment is a move in the right direction.
"We send £53m a day to Europe. We could do an awful lot with that money in terms of kickstarting growth and spending in the UK. We need that money here. Europe is dying on its feet," she said.
"If Cameron wanted to shoot the Ukip fox, the way to do it would be come out now and say 'I'm going to promise people a vote on EU membership now, before the next election.'
"If he did that, he would take away the reason for Ukip to exist."
Former chancellor Norman Lamont joined fellow Tory grandees Nigel Lawson and Michael Portillo by calling for Britain to exit the EU.
He said: "The advantages of the EU have been overstated and we could survive outside perfectly well.
"We should negotiate and see if we can a free trade agrerdemnt with the EU. If we can't get that we should leave."